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Poindron P, Piguet P, Förster E (eds): New Methods for Culturing Cells from Nervous Tissues. BioValley Monogr. Basel, Karger, 2005, vol 1, pp 1­11

Hippocampal Slice Cultures

Eckart Förster a, Marlene Bartosb, Shanting Zhaoa

a Institut für Anatomie und Zellbiologie I, bPhysiologisches Institut, Universität Freiburg, Freiburg, Germany

Introduction

Since the first description of organotypic hippocampal slice cultures based on the method of Gähwiler [1], more than two decades of practical experience have accumulated and make hippocampal slice cultures a wellcharacterized in vitro model to study the physiological properties of single neurons as well as neuronal circuits. In the following protocol the preparation of hippocampal slice cultures is described, where we incubate on millipore membranes according to the protocol described by Stoppini et al. [2]. Slice cultures prepared from the hippocampal region are likely the most widely used organotypic slice culture model of neural tissue. The hippocampus has a number of properties that predestinate this region of the cerebral cortex for the preparation of slice cultures: The organization of the hippocampus is much simpler than that of the neocortex. The principal neuronal cell types, pyramidal cells and granule cells, are easily identified and the neuronal connectivity of principal neurons within the hippocampus is well characterized. Interneurons are found in their typical positions. Neurons form autapses, therefore, the kinetic and dynamic properties of synaptic transmission can be studied at the single-cell level. The hippocampal region develops rather late when compared to the neocortex. Thus, the majority of the granule cells in the dentate gyrus are born in the early postnatal period. Therefore, numerous aspects of cortical development, such as neurogenesis, neuronal positioning, axonal pathfinding, neuronal specificity, synapse formation and network activity may be studied in hippocampal slice cultures prepared from early postnatal tissue. Hippocampal slices may also be cocultured with slices from other cortical areas. As an example, cocultures prepared from the entorhinal cortex and the hippocampus are a well-established in vitro model

to study the formation of axonal projections from the entorhinal cortex to the hippocampus [3­9]. A further advantage of organotypic slice cultures emerged with the frequent use of transgenic animals to address neuroscientific problems. The homozygous offspring of mutant mice sometimes does not survive more than a few hours or days after birth due to severe malfunctions in other organs than the brain. Thus, these defects preclude studies on the postnatal development of neural tissues, such as the hippocampal region. However, the immediate explantation of neural tissue after birth and the subsequent preparation of organotypic slice cultures allows to circumvent this problem since numerous aspects of postnatal hippocampal development (and some other brain regions) may also be studied in slice cultures [10]. In summary, hippocampal slice cultures are a suitable in vitro system to address a wide range of questions, concerning different aspects of neuronal development or function, such as axonal pathfinding [3­9], regeneration of fiber projections [4], cell migration [7], synaptic properties, network dynamics or the physiology of neuronal interactions [28].

Materials and Methods

Dissection Tools For the preparation of slice cultures, a suitable set of dissection tools is shown in figure 1: These dissection tools include two scalpels with small exchangeable blades, four to ten spatules, three fine scissors and three forceps with bend tips. Dissection tools should be autoclaved before the dissection procedure and are then placed in sterile glass or plastic dishes as shown in figure 1.

Media Usually two different media are used for the preparation, nutrition medium and preparation medium. Nutrition Medium Nutrition medium is composed of 25% heat-inactivated horse serum, 25% Hank's balanced salt solution, 50% minimal essential medium and 2 mM glutamine. By the addition of 580 l of 7.5% NaHCO3, a final concentration of 0.044% NaHCO3 is achieved. The pH is adjusted to 7.2. The media and supplements were purchased from Gibco/Invitrogen. Preparation Medium The composition of the preparation medium is basically the same as the nutrition medium; the only difference is the replacement of 25% serum by minimal essential medium. Serum is replaced since it is a rather expensive component, but not absolutely

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Fig. 1. Dissection tools required for the preparation of hippocampal slice cultures, including forceps, fine spatulas, scalpels with exchangeable blades and scissors.

required during the short preparation procedure (in principle, nutrition medium may also be used for the preparation).

Dissection Procedure For the preparation of hippocampal slice cultures, usually 0- to 7-days-old mouse- or rat pups are used. The experiments have to be performed in agreement with the institutional guide for animal care. After decapitation under hypothermic anesthesia, the skin is removed from the skull with two fine forceps. Then, one blade of a pair of fine scissors is introduced into the foramen magnum and the skull is opened by cutting along its caudal (back) to rostral (front) axis. Two cuts are then made perpendicular to the first one, the scissors pointing towards the left or right ear, respectively. The opening of the skull may then be carefully enlarged with fine forceps. By means of a spatule, the brain is then removed from the skull and transferred into a plastic dish containing ice-cold preparation buffer. It is recommended to place the dishes with the preparation buffer on a precooled metal block. Next, the cerebellum is removed from the explanted brain and the brain hemispheres are separated from each other with a scalpel (fig. 2a). It is recommended to perform the next steps of the procedure with two scalpels and under visual control by using a binocular. First, the brainstem is separated from the cortex (fig. 2b). During this step of the preparation, it has to be taken care not to damage the cortical tissue since the hippocampal region is located on the medial side of the hemisphere (for a three-dimensional view of the hippocampus location within the brain compare also with figure 2 in the chapter `Culture of Dissociated Hippocampal Neurons'). For the next step, the explantation of the hippocampus, a helpful landmark is a blood vessel that demarcates the border between the hippocampus and the adjacent cortical areas (fig. 2b, the upper black dotted line). The tissue is then cut along this blood vessel by means of a scalpel while the hemisphere is pinned to the culture plate with the help of the second scalpel. During the entire preparation it is important to perform clear cuts with the scalpel and to avoid separation of

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a

b

c Fig. 2. Preparation of hippocampal explants and slices, illustrated in four steps. a A brain hemisphere is shown after removal from the skull. The cortex (left) and the brain stem (right) are easily distinguishable. b The same hemisphere is shown after removal of the brain stem. The hippocampus is located between the black dotted lines. c Two hippocampi are shown, explanted from the two different cortical hemispheres of the same brain. For the preparation of slices, hippocampi are cut perpendicular to their length axis on a tissue chopper. Freshly prepared hippocampal slices are shown in (d ). Note the characteristic laminar structure of the hippocampal slices. The pyramidal cell layer and the dentate gyrus with the granule cell layer may be distinguished. Bars 200 m.

d

tissue pieces by simply stretching the tissue. This helps to preserve the integrity of the hippocampal tissue, including the characteristic neuronal connectivity which will be maintained to a certain extent within the hippocampal slice cultures. At this point, it is also possible to explant the hippocampus together with the adjacent entorhinal cortex for the preparation of complex slice cultures containing both a piece of entorhinal cortex and a hippocampal slice. The explanted hippocampus (fig. 2c) is then placed on a McIllwain tissue chopper (fig. 3) and cut perpendicular to its length axis. The razor blade, which is used for the slicing, should be cleaned with ethanol prior to use. The slice thickness is usually 300­400 m and may be adjusted with a micrometer screw. The hippocampus is then automatically cut into twenty to thirty slices, the number of slices depending on the adjusted thickness and on the age of the animal. By means of two humidified spatulas, the slices are transferred to a dish containing ice-cold nutrition medium (fig. 2d). The slices

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Fig. 3. McIllwain tissue chopper for the preparation of slices from neural tissue. For the preparation of hippocampal slices, an explanted hippocampus is placed on the circular support (left). The required slice thickness may be adjusted with the micrometer screw seen in the back. By means of the razor blade (left, above circular support) the hippocampus is then cut perpendicular to its length axis into slices (thickness usually 300­400 m).

are then carefully separated from each other and single slices are then carefully taken with the two spatulas and placed onto the membrane of a static tissue culture insert (Millipore) in a six-well plate containing 0.8­1.2 ml nutrition medium per well. Up to six slices may be placed onto a single Millipore membrane. The six-well plates with the tissue culture inserts are then transferred to an incubator with a humidified atmosphere containing 5% CO2 at 37 C. The medium is changed every 2 days. The required plastic ware may be purchased from Falcon.

Applications

Formation of Fiber Projections in Entorhino-Hippocampal Slice Cultures Defined axonal projections to the hippocampus, such as the perforant path, also form in vitro, in slice cultures. Thus, the coculture of a hippocampal slice and a slice from the entorhinal cortex is a well-characterized in vitro model to study the factors that control the formation of the perforant path [3­6]. Similarly, a commissural projection is formed when two hippocampal slices are cocultured together, thus allowing to study the factors that control the pathfinding of a commissural fiber projection in vitro [5, 9].

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Fig. 4. Laminar flow bench equipped for the preparation of organotypic slice cultures. A binocular for visual control of the preparation (center), a light source for the binocular (left), a tissue chopper (right), a precooled metal block with four dishes containing ice-cold preparation medium (front), dissection tools (behind), the two bottles on the right contain incubation medium or preparation medium, respectively.

Preparation of Entorhino-Hippocampal Cocultures and Hippo-Hippocampal Cocultures To study the formation of fiber projections from the entorhinal cortex to the hippocampus, entorhino-hippocampal cocultures were prepared, the slices containing the hippocampus and the adjacent entorhinal cortex of newborn mouse pups (P0). Using two spatulas, the complex entorhino-hippocampal slices were carefully placed onto membranes of Millicell-CM culture inserts (Millipore) and transferred to a six-well plate containing 1 ml/well nutrition medium (25% heat-inactivated horse serum, 25% Hank's balanced salt solution, 50% minimal essential medium, 2 mM glutamine, pH 7.2). These complex slices were cultivated as static cultures according to Stoppini et al. [2] for 12 days in vitro (DIV). In hippo-hippocampal cocultures the formation of a commissural projection from one slice to the second slice may be studied. Therefore, two hippocampal slices were placed in close vicinity on the membrane of tissue culture inserts and incubated as coculture as described above. Tracing of Entorhino-Hippocampal Fibers and Hippocampal Commissural Fibers After 10 DIV a crystal of biocytin (Sigma, Munich, Germany) was placed , on the entorhinal portion of the coculture in order to trace the entorhinal projection to the dentate gyrus (fig. 5). To trace the commissural projection in

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CA1

* *

EC

HIPPO

oml iml g

* *

CA3

Fig. 5. Formation of an entorhino-hippocampal fiber projection in a coculture of entorhinal cortex (EC) and hippocampus (HIPPO). To trace entorhinal fibers, biocytin was injected into the entorhinal slice (EC; injection sites are marked by asterisks). Note that the entorhinal fibers (black) form a sharp projection to the outer molecular layer (oml) of the dentate gyrus but do not invade the inner molecular layer (iml). g granule cell layer. The slice is counterstained with cresyl violet. CA1, CA3 pyramidal cell layer, CA1 and CA3 region, respectively. Bar 50 m.

hippo-hippocampal cocultures, a biocytin crystal was placed on the hilar region of one of the hippocampal slices, where the mossy cells are located, which give rise to the commissural axons. After 12 DIV the cultures were fixed with 4% para, formaldehyde, sectioned (50 m), and incubated with avidin-biotin-peroxidase complex (Vector Laboratories, Burlingame, Calif., USA). Sections were developed with diaminobenzidine/nickel and counterstained with cresyl violet. Enzymatic Digestion of Extracellular Matrix Components in Slice Cultures To study the role of the extracellular matrix component hyaluronan and molecules associated with hyaluronan in keeping axonal projections in their specific layers, some of the slices were incubated with the enzyme hyaluronidase (Calbiochem, San Diego, Calif., USA) during the incubation period. Hyaluronidase was diluted in 0.9% NaCl to concentrations ranging from 70­700 TRU/ml [8]. Two microliters of the diluted hyaluronidase was directly applied to each individual coculture two times per day, beginning with the day

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* *

gcl gcl

x hilus x

hilus 50 m 50 m

a

b Fig. 6. a Light-microscopical analysis of three neurons in a 42-day-old slice culture derived from an P0 mouse (C57/Bl6). Somata, dendrites and axonal arborizations are labeled. b Same cells as in a. Using the whole-cell patch-clamp recording technique (34 C), depolarizing currents were injected (700 pA) into the cells under current-clamp conditions. This resulted in a characteristic firing pattern which is shown for the neuron marked with a star and for the neuron labeled with an `x'. Neurons were labeled during the recordings with biocytin and subsequently processed for visualization (see text). gcl granule cell layer. Arrows point to axonal arbors.

of slice culture preparation until 10 DIV. Two microliters of 0.9% NaCl without enzyme was added to the control cultures. These experiments demonstrated that hyaluronan is required to keep entorhinal fibers in the outer molecular layer of the dentate gyrus. In contrast, laminar specificity of commissural fibers projecting into the dentate inner molecular layer does not depend on the extracellular matrix but on the position of the target cell [for details, see 8, 9].

Interneuron Identification in Hippocampal Slice Cultures

Cortical interneurons are diverse based on their morphological, physiological and neurochemical properties [11­25]. To determine if such diversity also exists in slice cultures, whole-cell patch-clamp recordings in combination with intracellular labelings have to be performed. Physiological characterization of interneurons is carried out under visual control using infrared differential interference contrast video-microscopy [26, 27]. Intrinsic physiological parameters (e.g., input resistance, membrane time constant and firing pattern) of the interneurons are determined in currentclamp conditions by injecting depolarizing and hyperpolarizing currents of certain duration and amplitude. For morphological identification, interneurons are filled during the experiments intracellularly with the cell marker biocytin and subsequently visualized

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hilus

gcl 50 m 50 m

a

b Fig. 7. a Light-microscopical analysis of a putative interneuron in a 42-day-old slice culture derived from an P0 mouse (C57/Bl6). Somata and dendrites are clearly labeled. b Same cell as in a. A recording technique was used as described in figure 6, to induce a train of action potentials in this neuron. Note the high-frequent firing of action potentials during depolarizing current injections (800 pA), which is typical for certain subtypes of interneurons. gcl Granule cell layer.

using avidin-biotinylated peroxidase complex and 3 ,3 -diaminobenzidinetetrahydrochloride as chromogen [28] (fig. 6). This approach is optimal for morphological three-dimensional reconstructions of the labeled cell using Neurolucida (MicroBrightField, Colchester, Vt., USA) to determine quantitative morphological parameters such as total dendritic and axonal length, or to develop single-cell passive cable models based on simulation environments such as NEURON [29] to study somato-dendritic processing of synaptic inputs. Biocytin-filled interneurons can also be visualized using avidin-conjugated fluorescent dyes (Alexa, Molecular Probes, Eugen, Oreg., USA). This labeling technique is optimal for immunohistochemical double-labeling with antibodies against certain interneuron-specific neurochemical markers such as Ca2 -binding proteins parvalbumin, calbindin, calretinin or neuropeptides such as somatostatin, cholecystokinin or vasoactive intestinal polypeptide [15]. Confocal image stacks can be used for the morphological identification of interneurons and to prove colocalization of interneuron-specific markers.

References

1 2 3 Gähwiler BH: Organotypic monolayer cultures of nervous tissue. J Neurosci Methods 1981; 4:329­342. Stoppini L, Buchs P-A, Muller D: A simple method for organotypic cultures of nervous tissue. J Neurosci Methods 1991;37:173­182. Li D, Field PM, Starega U, Li Y, Raisman G: Entorhinal axons project to dentate gyrus in organotypic slice co-culture. Neuroscience 1993;52:799­813.

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Li D, Field M, Yoshioka N, Raisman G: Axons regenerate with correct specificity in horizontal slice culture of the postnatal rat entorhino-hippocampal system. Eur J Neurosci 1994;6:1026­1037. Frotscher M, Heimrich B: Formation of layer-specific fiber projections to the hippocampus in vitro. Proc Natl Acad Sci USA 1993;90:10400­10403. Del Rio JA, Heimrich B, Borrell V, Förster E, Drakew A, Alcantara S, Nakajima K, Miyata T, Ogawa M, Mikoshiba K, Derer P, Frotscher M, Soriano E: A role for Cajal-Retzius cells and reelin in the development of hippocampal connections. Nature 1997;385:70­74. Förster E, Tielsch A, Saum B, Weiss KH, Johanssen C, Graus-Porta D, Müller U, Frotscher M: Reelin, Disabled1, and b1-class integrins are required for the formation of the radial glial scaffold in the hippocampus. Proc Natl Acad Sci USA 2002;99:13178­13183. Förster E, Zhao S, Frotscher M: Hyaluronan-associated adhesive cues control fiber segregation in the hippocampus. Development 2001;128:3029­3039. Zhao S, Förster E, Chai X, Frotscher M: Different signals control laminar specificity of commissural and entorhinal fibers to the dentate gyrus. J Neurosci 2003;23:7351­7357. Schwab MH, Bartholomae A, Heimrich B, Feldmeyer D, Druffel-Augustin S, Goebbels S, Naya FJ, Zhao S, Frotscher M, Tsai MJ, Nave KA: Neuronal basic helix-loop-helix proteins (NEX and BETA2/Neuro D) regulate terminal granule cell differentiation in the hippocampus. J Neurosci 2000;20:3714­3724. Amaral DG: A Golgi study of cell types in the hilar region of the hippocampus in the rat. J Comp Neurol 1978;182:851­914. Han Z-S, Buhl EH, Lorinczi Z, Somogyi P: A high degree of spatial selectivity in the axonal and dendritic domains of physiologically identified local-circuit neurons in the dentate gyrus of the rat hippocampus. Eur J Neurosci 1993;5:395­410. Sik A, Penttonen M, Ylinen A, Buzsáki G: Hippocampal CA1 interneurons: An in vivo intracellular labeling study. J Neurosci 1995;15:6651­6665. Ylinen A, Soltesz I, Bragin A, Penttonen M, Sik A, Buzsáki G: Intracellular correlates of hippocampal theta rhythm in identified pyramidal cells, granule cells, and basket cells. Hippocampus 1995;5:78­90. Freund TF, Buzsáki G: Interneurons of the hippocampus. Hippocampus 1996;6:347­470. Parra P, Gulyás A, Miles R: How many subtypes of inhibitory cells in the hippocampus? Neuron 1998;20:983­993. Mott DD, Turner DA, Okazaki MM, Lewis DV: Interneurons of the dentate-hilus border of the rat dentate gyrus: Morphological and electrophysiological heterogeneity. J Neurosci 1997;17: 3990­4005. Lübke J, Frotscher M, Spruston N: Specialized electrophysiological properties of anatomically identified neurons in the hilar region of the rat fascia dentata. J Neurophysiol 1998;79: 1518­1534. Gupta A, Wang Y, Markram H: Organizing principles for a diversity of GABAergic interneurons and synapses in the neocortex. Science 2000;287:273­278. Van Hooft JA, Giuffrida R, Blatow M, Monyer H: Differential expression of group I metabotropic glutamate receptors in functionally distinct hippocampal interneurons. J Neurosci 2000;20: 3544­3551. Emri Z, Antal K, Gulyás AI, Megias M, Freund TF: Electrotonic profile and passive propagation of synaptic potentials in three subpopulations of hippocampal CA1 interneurons. Neuroscience 2001;104:1013­1026. Bacci A, Rudolph U, Huguenard JR, Prince DA: Major differences in inhibitory synaptic transmission onto two neocortical interneuron subclasses. J Neurosci 2003;23:9664­9674. Klausberger T, Magill PJ, Márton PJ, Roberts JD, Cobden PM, Buzsaki G, Somogyi P: Brainstate- and cell-type-specific firing of hippocampal interneurons in vivo. Nature 2003;421: 844­848. Jonas P, Bischofberger J, Fricker D, Miles R: Interneuron diversity series: Fast in, fast outtemporal and spatial signal processing in hippocampal interneurons. Trends Neurosci 2004;27: 30­40. Monyer H, Markram H: Interneuron diversity series: Molecular and genetic tools to study GABAergic interneuron diversity and function. Trends Neurosci 2004;27:90­97.

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Stuart GJ, Dodt H-U, Sakmann B: Patch-clamp recordings from the soma and dendrites of neurons in brain slices using infrared video microscopy. Pflügers Arch 1993;423:511­518. Koh D-S, Geiger JRP, Jonas P, Sakmann B: Ca2 -permeable AMPA and NMDA receptor channels in basket cells of rat hippocampal dentate gyrus. J Physiol (Lond) 1995;485:383­402. Bartos M, Vida I, Frotscher M, Geiger JRP, Jonas P: Rapid signaling at inhibitory synapses in a dentate gyrus interneuron network. J Neurosci 2001;21:2687­2698. Hines ML, Carnevale NT: The NEURON simulation environment. Neural Comput 1997;9: 1179­1209.

Eckart Förster Institut für Anatomie und Zellbiologie I, Universität Freiburg DE­79104 Freiburg (Germany) Tel. 49 761 203 5058, Fax 49 761 203 5071, E-Mail [email protected]

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